1. Field of the Invention
The field of the invention is methods for inducing and detecting LDL receptor signaling.
2. Background of the Invention
The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for LDL receptor family members in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor proteins Disabled-1 (Dab1) and FE65 (2,3). Here, we disclose an extended set of cytoplasmic polypeptides that can participate in signal transmission by the LDL receptor gene family. Most of these novel polypeptides are adaptor or scaffold polypeptides that contain PID or PDZ domains and function in the regulation of cellular kinases, including tyrosine kinases, serine/threonine kinases (e.g. microtubule associated protein (MAP) kinases) and lipid kinases (e.g. PI kinases), cell adhesion, vesicle trafficking, or neurotransmission. We also show that binding of Dab1 competes with receptor internalization indicating a mechanism by which signaling through this class of receptors might be regulated.
The invention provides methods and compositions for detecting and modulating, including inducing and suppressing, signal transduction through LDL receptors.
In a particular embodiment, the methods involve specifically detecting a stress that alters a functional interaction of an LDL receptor binding polypeptide with an LDL receptor interaction domain, the method comprising steps (a) introducing a predetermined stress into a system which provides a physical interaction of an LDL receptor binding polypeptide with an LDL receptor intracellular binding polypeptide interaction domain, whereby the system provides a stress-biased interaction of the binding polypeptide and the interaction domain, wherein the absence of the stress, the system provides an unbiased interaction of the binding polypeptide and the interaction domain; and (b) detecting the stress-biased interaction of the binding polypeptide and the interaction domain, wherein a difference between the stress-biased and unbiased interactions indicates that the stress alters the interaction of the binding polypeptide and the interaction domain. The binding polypeptide is independently selected from SEMCAP-1, JIP-1, PSD-95, JIP-2, Talin, OMP25, CAPON, PIP4,5 kinase, Na channel brain 3, Mint1, ICAP-1 and APC subunit10; and the receptor may be selected from very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor-2 (ApoER2), low density lipoprotein receptor (LDLR), low density lipoprotein receptor related protein (LRP), MEGF7 and Megalin. In panicula embodiments, the binding polypeptides and receptors are of natural sequence, preferably human sequence. In particular embodiments, the system is a cell expressing both the binding polypeptide and the interaction domain or an in vitro, cell-free mixture comprising a determined amount of the binding polypeptide and the interaction domain; exemplary systems include two-hybrid, biochemical pull-down, fluorescent polarization and solid phase binding assays.
The compositions of the invention, useful in the subject methods, include the subject binding polypeptides and mixtures consisting essentially of an LDL receptor binding polypeptide and an LDL receptor interaction domain, wherein the receptor may be independently selected from VLDLR, ApoER2, LDLR, LRP, MEGF7 and Megalin. Other aspects of the invention include nucleic acids encoding the disclosed binding polypeptides, antibodies which specifically bind them, and methods of use.